Use of poly-α2,8-sialic acid mimetic peptides to modulate NCAM functions

ABSTRACT

The invention relates to the use of a peptide consisting of 5 to 30 amino acid residues, preferably 9 to 15, most preferably about 12 amino acid residues, said peptide comprising a B epitope of a poly-α2,8 sialic acid attached to NCAM, which is recognized by an anti-poly-α2,8 sialic acid (PSA) antibody, for the preparation of a medicament for modulating NCAM functions, to be administered for the prevention and/or the treatment of neurodegenerative diseases, brain and spine lesions, age-related learning and memory problems, and cancer.

The invention relates to the use of poly-α2,8-sialic acid (PSA) mimeticpeptides to modulate specifically PSA-dependent NCAM functions in vitroand in vivo, and to their application for the treatment ofneurodegenerative diseases, brain and spine lesions, age-relatedlearning and memory problems, and cancer.

The ability of cell to modify its cell surface interactions with othercells, including neurons and glial cells, is a critical component ofnervous tissue development, remodelling and repair, as well as tumorformation and metastasis. Among many candidate molecules that arepotentially involved in such a process, isoforms of the neural celladhesion molecule (NCAM), a member of the IgG super-family, carrying anunconventional carbohydrate polymer, poly-α2,8-sialic acid (PSA), are ofparticular interest.

PSA is a polymer of negatively charged N-acetylneuraminic acid (sialicacid) residues in an alpha 2,8 linkage. Single PSA chain carried byNCAM, consists generally of at least 30 repeating units with the chainlength varying substantially in NCAM isolated from various sources (vonDer Ohe et al., Glycobiology, 2002, 12, 47-63; Rougon et al., Eur. J.Cell. Biol., 1993a, 61, 197-207). By comparison, poly-α2,8-sialic acidwhich is found in the capsule of bacteria such as Neisseria meningitidisGroup B and E. coli K1 forms longer polymers of about 200 repeatingunits. Studies using NMR microscopy indicate that PSA has a helicalstructure in solution consisting of eight or more contiguous sialic acidunits (Rougon et al., 1993a, precited; Yamasaki et al. 1991). PSA has alarge hydrated volume and high negative charge density, and therefore iswell placed to attenuate adhesion forces and to negatively regulateoverall cell surface interactions (Rutishauser et al., Science, 1988,240, 53-57). All the known alternatively spliced isoforms of NCAM can bepolysialized at the fifth Ig-like domain (Rougon et al., 1993 a,precited) and NCAM is the only clearly identified carrier of polysialicacid in the nervous system (Rougon et al., J. Cell. Biol., 1986, 103,2429-2437). Although, there is one report describing the association ofPSA with the α chain of the Na channel (Zuber et al J. Biol. Chem.,1992, 267, 9965-9971), the absence of PSA immunoreactivity in NCAMknock-out mice (Cremer et al., Nature, 1994, 367, 455457) suggests thatNCAM is the major if not the only carrier of PSA in vertebrate brain.

The attachment of PSA to NCAM is a developmentally regulated process;NCAM with high PSA content is associated with morphogenetic changesduring development such as cell migration, synaptogenesis and axonalgrowth and branching, while in adult brain poorly sialylated forms ofNCAM are dominating (Rougon et al., Polysialic Acid, 1993b, Roth J. R.,Rutishauser U. and Troy F. A. (eds), Birkhauser-Verlag: Basel, 323-333;Rutishauser et al, 1998, precited; Edelman et al., Annu. Rev. Cell.Biol., 1986, 2, 81-116). However, PSA-NCAM does persist in adult brainstructures that display a high degree of plasticity (Rougon et al.,1993a, precited). For example, PSA-NCAM is required for two essentialforms of activity-induced synaptic plasticity, long-term potentiation(LTP) and long-term depression (LTD), that are believed to be central tolearning and memory as well as activity-dependent pattern formationduring development. (Muller et al, Neuron, 1996, 17, 413-422). Indeed,hippocampal tissue prepared from the NCAM mutant mice exhibited amarkedly reduced capacity for LTP as well as LTD and this defect couldbe mimicked by the enzymatic destruction of the PSA moiety of NCAM.These observations indicate that PSA rather than the NCAM protein isrequired for plasticity. Morphological plasticity occurring in thehypothalamo-neurohypophyseal system (Theodosis et al., J. Neurosci.,1999, 19, 10228-10236) is also dependent upon the presence of PSA as invivo injection of endoneuraminidase prevents it.

PSA-NCAM is re-expressed in several pathological situations such asmuscle regeneration, axonal regeneration and brain tumors(Figarella-Branger et al., Cancer Res., 1990, 50, 6364-6370; Dubois etal., Neuromuscul. Disord., 1994, 4, 171-182; Aubert et al., Comp.Neurol., 1998, 399, 1-19; Muller et al., Neuroscience, 1994, 61,441-445) or brain neurodegenerative diseases (Le Gal La Salle et al., J.Neurosci., 1992, 12, 872-882). Based on these observations, PSA-NCAMemerged as an important permissive factor for dynamic changes in cellsurface interactions required for morphogenesis and tissue remodelling(Rougon et al., 1993b, precited; Figarella-Branger, 1993; Rutishauser,Development, 1992, 99-104).

Many tumors with neural and endocrine characteristics expressedPSA-NCAM. For example, PSA-NCAM had been detected in neuroblastomas andmedulloblastomas (Figarella-Branger et al., precited), small cellcarcinoma of the lung (Patel et al., Int. J. Cancer, 1989, 44, 573-578)and rhabdomyosarcomas, and is possibly related to the invasive andmetastatic potential of these tumors (Rougon et al., 1993b, precited).Recently, injection of neuraminidase into a nude mouse model formetastasis showed that removal of PSA on the primary tumor delayedmetastasis. (Daniel et al, Oncogene, 2001, 20, 997-1004).

Thus, the molecule PSA-NCAM and more precisely the carbohydrate PSArepresents one of the potential targets of future therapeutic approachesto promote plasticity and functional recovery after brain damage or toprevent metastasis formation.

Therefore, several strategies have been developed to modulate PSAfunctions:

-   -   genetic manipulations: NCAM or polylsialyltransferase knock-out        mice (Cremer et al., precited): this strategy does not open        perspective for therapy,    -   enzymatic digestion: endoneuraminidase (Theodosis et al.,        precited; Daniel et al., precited): its therapeutic potential is        rather limited owing to its large size and restricted diffusion        in vivo and to the possibility of inducing an immune response,    -   anti-PSA monoclonal antibodies (Monnier et al., Developmental        Biology, 2001, 229, 1-14); their therapeutic potential is rather        limited owing to their antibody nature,

Colominic acid, a PSA analog isolated from bacterial capsule; itstherapeutic potential is rather limited owing to its instability atacidic pH and to the impossibility to control its exact composition interms of purity and homogeneity (calibration of the sialic acid chainlength, from one batch to another),

-   -   N-butanoylmannosamine (ManBut), a small molecule capable of        inhibiting PSA biosynthesis in vitro (Mahal et al., Science,        2001, 294, 380-382); its activity has not been demonstrated in        vivo.

However, to date these strategies which have been used to uncovermechanisms of action or functions of PSA, have not led to the discoveryof new drugs able to modulate PSA functions in vivo.

Therefore, there is a need for new molecules able to modulatespecifically PSA-dependent NCAM functions in vivo, which can be used aspharmaceutical compositions for promoting plasticity and functionalrecovery after brain or spine damage or for preventing metastasis.

Peptides representing molecular mimetics of carbohydrate epitopes frommicroorganisms including Neisseria meningitidis group B PSA-specificepitopes have been described in view of developing safe and efficientvaccine candidate against these microorganisms (Shin et al., Infectionand Immunity, 2001, 69, 3335-3342); the Neisseria meningitidis group BPSA-specific peptides disclosed in Shin et al., represent epitopes whichare different from vertebrate cells (neurons) PSA epitopes and thus donot induce antibodies which bind to said neuronal PSA (PSA attached toNCAM) and may cause neurological damage. In addition, Shin et al.disclose only two peptides (DHQRFFV (SEQ ID NO: 31) and AHQASFV (SEQ IDNO: 32) representing mimetics of PSA-attached to NCAM epitopes, whichare used as control to demonstrate the specificity of Neisseriameningitidis group B PSA mimetic peptides.

The inventors have isolated other peptides which are molecular mimeticsof PSA-attached to NCAM epitopes and they have demonstrated that thesePSA mimetic peptides are able to modulate (enhance or inhibit), in vivo,in a PSA-dependent manner, cellular processes that are normally affectedby polysialylation of NCAM. For example, the inventors have demonstrateda significant effect of the PSA mimetic peptides, in vivo, on axonsgrowth, guidance and fasciculation as well as on neurons migration.Moreover, the administration of the PSA mimetic peptides results infunctional recovery after spinal cord injury and is also accompanied byreduced reactive gliosis at the site of the lesion.

In the context of the invention, poly-α2,8 sialic acid or PSA meansPSA-attached to NCAM, as opposed to other PSA such as PSA from bacteriacapsules.

These PSA mimetic peptides which are active in vivo at low doses (μMconcentration) and do not exhibit any cytotoxicity are useful for:

-   -   the treatment of neurodegenerative diseases (Parkinson,        Huntington's chorea, Alzheimer, multiple sclerosis): they are        useful as adjuvants for cell therapy; graft of neuronal        progenitors (expressing PSA) in combination with PSA mimetic        peptides would enhance significantly progenitors migration and        axon outgrowth in the damaged areas of the brain,    -   the treatment of brain and spine lesion: they would promote        functional recovery after brain or spine damage by increasing        axon survival and regeneration,    -   the prevention and treatment of age-related learning and memory        problems: they would promote nervous system plasticity by        increasing neurogenesis and/or synaptic plasticity,    -   the treatment of cancer: they would prevent metastasis formation        by inhibiting cell migration from PSA expressing tumors, for        example neuroectodermic tumors.

In addition to said therapeutic use, the PSA mimetic peptides are usefulas complementary tools to uncover mechanisms of action and unknownfunctions of the carbohydrate PSA.

The present invention relates to the use of a peptide consisting of 5 to30 amino acid residues, preferably 9 to 15, most preferably about 12amino acid residues, said peptide comprising a B epitope of a poly-α2,8sialic acid attached to NCAM, which is recognized by an anti-poly-α2,8sialic acid antibody, for the preparation of a medicament for modulatingNCAM functions.

The “B epitope recognized by an anti-PSA antibody” refers to a peptidewhich reacts specifically and selectively, in vitro and in vivo, withthe paratope of an antibody produced by lymphoid cells in response to astimulation with poly-α2,8 sialic acid as an immunogen. Anti-PSAantibodies prepared by standard techniques, following protocols asdescribed for example in Antibodies: A Laboratory Manual, E. Howell andD Lane, Cold Spring Harbor Laboratory, 1988 are well-known in the art;they include, without limitation, monoclonal antibodies 735 (Frosch etal., P.N.A.S; 1985, 82, 1194-1198), 30H12 (Coquillat et al., Infect.Immun., 2001, 69, 7130-7139) or MenB (ABCYS AbC0019; Rougon et al., J.Cell. Biol., 1986, 103, 2429-2437).

The invention includes the use of linear and cyclic peptides. Preferredcyclic peptides as defined in the invention comprise peptides in whichthe side chain of one amino acid in the peptide chain is attachedcovalently to the side chain of another amino acid in the peptide chainvia formation of a covalent bond, such as a disulfide bond between twocysteine residues.

The peptides as defined in the invention refer to peptides which havethe following activities:

-   -   antibody binding activity: they are recognized specifically by        an anti-PSA monoclonal or polyclonal antibody, and    -   biological activity: they have the activity of modulating        (enhancing or inhibiting) PSA-dependent NCAM functions.

The peptides as defined in the invention are denominated hereafter PSAmimetic peptides, mimetic peptides or peptides. Unless otherwisespecified, said peptides are either linear or cyclic.

The mimetic peptides antibody-binding activity is verified by standardimmunoassay which are well-known by a person skilled in the art; forexample, 100% peptide binding is observed at concentrations of 10⁻⁴M andabove in an ELISA with an anti-PSA monoclonal antibody at theconcentration of 2.5 μg/well, and this antibody binding activity isspecifically inhibited by a PSA analog such as colominic acid.

The mimetic peptides biological activity is verified by standard cellgrowth and cell migration assays in vitro or in vivo, which arewell-known by a person skilled in the art; for example, assays onprimary neurons from different sources (dorsal root ganglion, cerebellarneurons, retina, . . . ) show the following effects:

-   -   a significant dose dependent increase of neurite length in vivo        and in vitro,    -   a significant effect on axonal guidance in vivo and in vitro        (axons defasciculation), and    -   a significant increase or inhibition of cell migration.

In addition, functional assays including study of the functionalrecovery from spinal cord hemisection by well-known tests (BBB test:Basso, Beattie and Bresnahan test; Basso et al., Restor. Neurol.Neurosci, 2002, 5, 189-218; rotarod test) show a significant locomotorand fine motor coordination recovery in the mimetic peptide treatedanimals, by comparison with the animals treated with a non relevantpeptide.

The mimetic peptide biological activity is specifically inhibited byenzymatic digestion with endoneuraminidase and is absent in NCAMknock-out mice.

According to an advantageous embodiment of the use of the invention,said peptide comprises a sequence which is selected from the groupconsisting of the sequences SEQ ID NO: 1 to 12 and 14 to 26,corresponding respectively to DSPLVPFIDFHP, LWQPPLIPGIDF, QIEPWFTPEDFP,TRLAPLVFPLDY, SWLQMPWALVRT, EIHLRMIKQITI, WHLEYMWRWPRL, LIEQRLPKHILT,YETSSSRLLAYA, TLASQLSNTSAY, SDQGVNGSWSNP, WHNWNLWAPASPT, IKSPLTWLVPPD,SHLDLSTGHRTS, CYPLNPEVYHCG, CWPLSHSVIVCG, CSSVTAWTTGCG, CYMASGVFLCG,CWPLGPSTYICG, CSLIASMETGCG, CSKIASMETGCG, CYIGDPPFNPCG, CWPLGDSTVICGCPLRLAFTFGCG and CTRMSHGYWICG.

According to another advantageous embodiment of the use of theinvention, said peptide is a linear peptide comprising the sequenceWHWQWTPWSIQP (SEQ ID NO: 13).

According to another advantageous embodiment of the use of theinvention, said peptide is a cyclic peptide comprising the sequenceWHWQWTPWSIQP (SEQ ID NO: 13).

The invention also includes the use of any functional derivative of thepeptides as defined above, comprising one or more modifications which donot affect substantially the antibody binding and biological activitiesof the initial peptide.

Such modifications include for example: addition and/or deletion and/orsubstitution of one or more amino acid residue in the peptide chain,and/or replacement of one or more of the amide bond by a non-amide bond,and/or replacement of one or more amino acid side chain by a differentchemical moiety, and/or protection of the N-terminus, the C-terminus, orone or more of the side chain by a protecting group, and/or introductionof double bonds and/or cyclization and/or stereo-specificity into theamino acid chain to increase rigidity, and/or binding affinity and/orenhance resistance to enzymatic degradation of the peptides. Since allthe variations are known in the art, it is submitted that a personskilled in the art will be able to produce, test, identify and selectother peptides/epitopes according to the present invention.

For instance, it is possible to substitute amino acids by equivalentamino acids. “Equivalent amino acid” is used herein to name any aminoacid that may substitutes for one of the amino acids belonging to theinitial peptide structure without modifying the antibody binding andbiological activities of the initial peptide structure. These equivalentamino acids may be determined by their structural homology with theinitial amino acids to be replaced and by their biological activity onthe target cells of the peptides according to the invention. As anillustrative example, it should be mentioned the possibility of carryingout substitutions like, for example, leucine by valine or isoleucine,aspartic acid by glutamic acid, glutamine by asparagine, asparagine bylysine etc., it being understood that the reverse substitutions arepermitted in the same conditions. In some cases, it may also be possibleto replace of a residue in the L-form by a residue in the D-form or thereplacement of the glutamine (Q) residue by a pyro-glutamic acidcompound.

Preferably, said peptide consists of a sequence selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 26.

Most preferably, said peptide is selected from the group consisting of:

-   -   a linear peptide presenting SEQ ID NO: 1 (DSPLVPFIDFHP)        denominated hereafter p21, and    -   a cyclic peptide in which the side chain of the cysteine residue        at position 1 of SEQ ID NO: 18 (CSSVTAWTTGCG) or SEQ ID NO: 22        (CSKIASMETGCG) is attached covalently to the side chain residue        of the cysteine at position 11 of SEQ ID NO: 18 or SEQ ID NO: 22        via a disulfide bond; said peptides are denominated hereafter,        respectively p65 and p66.

According to another advantageous embodiment of the use of theinvention, said peptide is associated with another peptide ornon-peptide molecule and/or incorporated into a suitable supportincluding for example, polymers, lipidic vesicles, microspheres,proteins and the like. Preferably, the other peptide or nonpeptidemolecule and/or the support as defined above enables the mimetic peptideto cross the brain-blood barrier.

Such association which may improve the peptide solubility, absorption,bioavailability, biological half life, is formed, by using techniqueswell known in the art; it may be through, without limitation, covalentbonding (e.g., amide bond, disulfide bond . . . ), or through chelation,electrostatic interactions, hydrophobic interactions, hydrogen bonding,ion-dipole interactions, dipole-dipole interactions, or any combinationof the above.

According to another advantageous embodiment of the use of theinvention, said peptide is incorporated in a complex comprising aplurality of identical or different peptides according to the invention,linked by covalent or non-covalent bonds.

According to another advantageous embodiment of the use of theinvention, said peptide is associated with a marker such as afluorescent marker, to facilitate the detection of the peptidesaccording to the invention.

According to another advantageous embodiment of the invention, saidpeptide is included in a fusion protein to allow expression of saidpeptide.

According to another advantageous embodiment of the use of theinvention, said medicament is for the prevention and/or the treatment ofa pathological condition selected from the group consisting of:neurodegenerative diseases, brain and spine lesions, age-relatedlearning and memory problems.

According to another advantageous embodiment of the use of theinvention, a peptide selected from the group consisting of: the linearand the cyclic peptides comprising or consisting of the sequences SEQ IDNO: 1 to 12 and 14 to 26 and the cyclic peptides comprising SEQ ID NO:13, or a complex thereof as defined above, is used for the preparationof a medicament for the prevention and/or the treatment of cancer.

The peptides as defined in the present invention may be prepared by anysuitable process. Preferably, it is obtained by chemical synthesis inliquid or solid phase by successive couplings of the different aminoacid residues to be incorporated (from the N-terminal end to theC-terminal end in liquid phase, or from the C-terminal end to theN-terminal end in solid phase) wherein the N-terminal ends and thereactive side chains are previously blocked by conventional groups. Forsolid phase synthesis the technique described by Merrifield (J. Am.Chem. Soc., 1964, 85, 2149-2154) may be used.

The peptides as defined in the present invention may also be obtained bygenetic engineering technology. A typical example comprise culturing ahost cell containing an expression vector comprising a nucleic acidsequence encoding said peptide, under conditions suitable for theexpression of the peptide, and recovering the peptide from the host cellculture. The peptide may be included in a fusion protein by cloning acDNA into an expression vector in frame with a polynucleotide coding forthe peptide of the invention. Alternatively, multimer of identical ordifferent peptides can also be produced by expressing a polynucleotidecoding for multiple copies of a monomer, or coding for differentmonomers.

The invention further concerns a medicament comprising a peptide or apeptide complex as defined above, with the exclusion of the linearpeptide comprising the sequence SEQ ID NO: 13.

The invention further concerns a pharmaceutical composition comprisingan effective amount of a peptide or a peptide complex as defined above,with the exclusion of the linear peptide comprising the sequence SEQ IDNO:13, in a combination with a pharmaceutically acceptable carrier.

The carriers of the pharmaceutical compositions of the invention can beany vehicle for parenteral, intrathecal, oral, aerosol, nasal, or ocularadministration of drugs acting on the nervous system. For example, acomposition according to the invention is administered intrathecallywhich enables the penetration of the composition directly into theCentral Nervous System. Alternatively, it is administered through thenose which enables the penetration of the aerosol composition to theCentral Nervous System through the olfactory nerve, or via the ocularroute, or by any other suitable method of administration as described inW. M. Pardridge, Peptide drug Delivery, Raven Press, N.Y., 1991.

The amount of peptide in the composition is in a concentration rangesfrom about 0.1 μM to about 10 μM. The preferred frequency ofadministration and effective dosage will vary from one subject toanother.

The invention also concerns a peptide as defined above, consisting of 5to 30 amino acid residues, preferably 9 to 15, most preferably about 12amino acid residues, said peptide comprising a B epitope of a poly-α2,8sialic acid attached to NCAM, which is recognized by an anti-poly-α2,8sialic acid (PSA) antibody, with the exclusion of the linear peptidescomprising the sequences selected in the group consisting of:WHWQWTPWSIQP (SEQ ID NO: 13); DHQRFFV (SEQ ID NO: 31) and AHQASFV (SEQID NO: 32).

The invention also provides a polynucleotide encoding said peptideaccording to the invention, as well as the complement of saidpolynucleotide, and fragments of at least 5 nucleotides thereof.

In particular, the invention provides the nucleotide sequences encodingthe peptides SEQ ID NO: 1 to 12 and SEQ ID NO: 14 to 26, including allpossible examples of nucleotide sequences encoding these peptides whichresult from the degeneration of the genetic code.

Nucleic acids of the invention may be obtained by the well-known methodsof recombinant DNA technology and/or chemical DNA synthesis.

The invention also provides recombinant vectors comprising apolynucleotide encoding a peptide of the invention. Vectors of theinvention are preferably expression vectors, wherein a sequence encodinga peptide of the invention is placed under control of appropriatetranscriptional and translational control elements. These vectors may beobtained and introduced in a host cell by the well-known recombinant DNAand genetic engineering techniques.

The invention also comprises a prokaryotic or eukaryotic host celltransformed by a vector of the invention, preferably an expressionvector.

The peptides as defined in the invention have the following advantages:

-   -   they are active in vivo at low doses (0.5 μM concentration),    -   they are stable in vivo,    -   they are very efficient in vivo since they act extracellularly;        thus, their activity is not limited by their ability to        penetrate inside the cell,    -   they are not toxic, and    -   they can be produced easily in large quantities.

The present invention will be further illustrated by the additionaldescription and drawings which follow, which refer to examplesillustrating the isolation, the binding specificity and the biologicaleffects of the PSA mimetic peptides according to the invention. Itshould be understood however that these examples are given only by wayof illustration of the invention and do not constitute in anyway alimitation thereof.

FIG. 1 illustrates PSA-NCAM structure.

FIG. 2 illustrates the effect of PSA mimetic peptides p65 and p66, onneurite outgrowth in vitro: mouse dorsal root ganglion explants (E13,5)were cultured in absence (A, D) or presence of the p65 and p66 peptidescoated on the microplates as BSA conjugate (B,E) or under soluble form(C,F). (G) quantification of the effect of the mimetic peptides on themean length of the longest neurite. *** P<0.001 compared with controlwith Student's t test. (H) cumulative frequency distribution plot of themean length of the longest neurite.

FIG. 3 illustrates the effect of PSA mimetic peptide p65 onfasciculation and guidance in vivo: E9 chick whole retina mountedpreparation (A) and its schematic drawing demonstrating the position ofthe DiI crystal (B). The dashed rectangle indicates the area from whichphotographs were taken. Arrows point toward the optic fissure. Axons ofE9 chick retina injected at E3 with the reverse peptide (C,F) or the p65peptide (D,G,E,H). Arrowheads show examples of axons that leave theirfascicle and run perpendicularly to it.

FIGS. 4 to 7 illustrate the effect of PSA mimetic peptides on cellmigration in vitro, analysed on subventricular zone explants (P1) fromnormal mice (NCAM +/+), heterozygous mice (NCAM +/−) or knock-out mice(NCAM −/−), cultured in presence of the mimetic peptides (p65, p21,p66), different form of said peptides (cyclic, linear, linear andacetylated), the control peptides (reverse p65 and reverse p66 and p22)or endosialidase N:

-   -   FIG. 4 illustrates the effect of the reverse p65 peptide (A,D),        endosialidase N (B,E) or the mimetic peptides p65 and p66        (C,F). (G) quantification of the effect of the mimetic peptides        on the cell migration mean distance. *** P<0.001 compared with        control with student's t test. (H) cumulative frequency        distribution plot of the mean distance of migration. (I)        Dose-dependant effect of the p65 peptide on the mean distance of        cell migration,    -   FIG. 5 illustrates the effect of different forms of p65 mimetic        peptide (cyclic, linear, linear and acetylated) on the cell        migration mean distance, compared with reverse p65 control        peptide. *** P<0.001 compared with control with student's t        test,    -   FIG. 6 illustrates the effect of p65 mimetic peptide on the cell        migration mean distance in knock-out mice (SCAM −/−) or        heterozygous mice (NCAM +/−). Endo N treated cells from NCAM        +/−mice and reverse p65 treated cells are included for        comparison. *** P<0.001 compared with control with student's t        test, and    -   FIG. 7 illustrates the effect of p65 and p21 mimetic peptides on        the cell migration mean distance. Endo N treated cells and cells        treated with control peptides (reverse p65 and p22) are included        for comparison. *** P<0.001 compared with control with student's        t test.

FIG. 8 illustrates the effect of PSA mimetic peptide p65 on cellmigration in vivo: (A) schematic drawing of the transplantation. (B)Confocal microscopy photography of a section showing the RMS (RostralMigration Stream) of grafted mice in presence of p65 peptide. Arrowheadsshow examples of GFP and PSA positive cells. SVZ explants (P1) weregrafted in presence of the reverse peptide (C,D) or the p65 peptide(E,F) and brains were analysed three days after graft (C,E) or four dayafter graft (D,F). (G) quantification of the effect of the p65 peptideon the number of GFP positive cells that reach the olfactory bulb threedays after graft. * P<0.05 compared with control with Student's t test.

FIG. 9 illustrates the protocol used to analyze the functional recoveryfrom spinal cord injury after injection of p65 peptide or p65 reversepeptide taken as control.

FIG. 10 illustrates the functional recovery from spinal cord injuryafter injection of p65 peptide (p65) or p65 reverse peptide (Rev) takenas control. A: Basso, Beattie and Bresnahan test (BBB test). B: Rotarodtest n=11 for p65 and n=8 for Rev. *** P<0.01, ** P<0.01, * P<0.05,compared with control with Student's t test.

FIG. 11 illustrates the decrease of reactive gliosis after spinal cordinjury, in mice treated with p65 peptide or p65 reverse peptide taken ascontrol. A: Immunofluorescence analysis using anti-GFAP and anti-PSAantibodies alone or in combination (double-labelling). B: quantificationof the GFAP staining. * P<0.05, compared with control with Student's ttest.

FIG. 12 illustrates the binding specificity of the cyclic mimeticpeptides:

FIG. 12A: ELISA using 30H12 anti-PSA monoclonal antibody-coated plates.Numbers 1 to 16 correspond to the peptide sequences as presented inTable IV.

FIGS. 12B, 12C and 12D: competitive binding to PSA-NCAM expressingcells. B: pre-incubation of p65 (1 mM) or p66 (1 mM) with MenB anti-PSAantibody. C: 30H12 anti-PSA antibody without peptide. D: pre-incubationof p65 (1 mM) or p66 (1 mM) with 30H12 anti-PSA antibody.

EXAMPLE 1 Peptide Library Screening with Anti-PSA Monoclonal Antibody

1) Materials and Methods

1.1) Materials

Peptide 12-mer Phage Display Library

Two libraries were screened. The first library (12™ Phage Displaypeptide Library, NEW ENGLAND BIOLABS) comprises 12-mer linear peptidespresented on the surface of M13-like phage particles as fusion proteinto the N-terminus of the pIII minor coat-protein (5 copies/phageparticle). The variance of the library differs from 10⁸ to 10⁹ peptideswith constant sequence length.

The second library, prepared as described in Felici et al. (J. Mol.Biol., 1991, 222, 301-301), comprises 12-mer cyclic peptides including 2cysteine residues at position 1 and 11 linked by a disulfide bond,presented on the surface of M13-like phage particles as fusion proteinto the N-terminus of the pVIII major coat-protein (100 copies/phage).The library comprises approximately 10⁸ peptides with constant sequencelength.

Anti-PSA Monoclonal Antibody (mAb).

Anti-PSA monoclonal antibodies, prepared by standard techniques asdescribed in Antibodies: A Laboratory Manual, E. Howell and D Lane, ColdSpring Harbor Laboratory, 1988, are used. For example, monoclonalantibodies 735 (Frosch et al., P.N.A.S; 1985, 82, 1194-1198), 30H12 (IgG2a; Coquillat et al., Infect. Immun., 2001, 69, 7130-7139) or MenB (IgM;ABCYS AbC0019) may be used.

-   -   plates (Maxisorp™ NUNC)    -   tubes (Maxisorp™, NUNC)    -   E. coli strain ER2537 (NEW ENGLAND BIOLABS)    -   96 gIII sequencing primer (NEW ENGLAND BIOLABS):

5′CCCTCATAGTTAGCGTAACG-3′ (SEQ ID NO: 27)

1.2) Buffers

-   -   Blocking solution: 0.5% BSA in PBS    -   Coating solution: 25 μg/ml anti-PSA mAb in PBS    -   TBS: 50 mM Tris-HCl pH 8.6, 150 mM NaCl.    -   TBST: TBS containing 0.1% or 0.5% Tween 20.    -   PEG /NaCl: 20% (W/V) polyethylene glycol-8000, 2.5 M NaCl.    -   Iodide Buffer: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 4M NaCl.

1.3) Method

Maxisorp™ tubes were incubated overnight with 2 ml of coating solution,at +4° C., with gentle agitation, according to the manual of the Ph.D.12™ Phage Display peptide Library kit (NEW ENGLAND BIOLABS). The coatingsolution was removed and used for the coating of new tubes for newrounds of biopanning. The coated tubes were incubated with 2 ml ofblocking solution for 1 h and washed 6 times with TBST. The tubes werefilled with 2 ml of phage solution (7.5 10¹⁰ pfu/ml in TBST containing0.1% Tween 20) and incubated with gentle agitation at room temperaturefor 1 h. After removal of the phage solution, the tubes were washed 10times with TBST. Bound phages were either eluted, specifically with 1 mMcolominic acid in TBS for 1 h, or non-specifically with 0.2 M glycineHCl (pH 2.2) for 10 min with immediate neutralization with 1 M Tris-HCl.Eluates were amplified in 20 ml of E. coli ER2537 culture (startingOD₆₀₀: 0.03), for 4.5 h at 37° C. with vigorous shaking. The cultureswere centrifuged for 10 min at 10,000 rpm at 4° C. The supernatant wastransferred to a fresh tube and centrifuged for another 10 minutes.PEG/NaCl solution was added to the supernatant (1 volume PEG/NaCl for 6volumes supernatant) and phage was precipitated overnight at 4° C. Thesolution containing the precipitate was centrifuged for 15 min at 10,000rpm at 4° C. The supernatant was decanted and the pellet was suspendedin 1 ml TBS and re-precipitated with ⅙ volume of PEG/NaCl for 1 h onice. After centrifugation, the pellet was finally suspended in 200 mlTBS, 0.02% NaN₃.

This amplified eluate was dissolved in TBST and a second and a thirdround of biopanning were carried out as described above; for the secondround, the TBST used for washing and phage incubation contained 0.1%Tween 20; in the third round the content was 0.5%.

The non-amplified eluate from the third round was subsequently titteredon LB/IPTG/X-gal plates. Blue plaques were picked and the phage cloneamplified in 2 ml E. coli ER2537 culture for 4.5 h at 37° C., withvigorous shaking. After centrifugation for 10 min at 10,000 rpm, at 4°C., the supernatant was mixed with ⅙ volume PEG/NaCl and the phageprecipitated at 4° C. overnight. The precipitate was centrifuged for 15min at 10,000 rpm at 4° C. The pellet was suspended in 100 μl TBS. 10 μlfrom this solution were mixed with 100 μl iodide buffer and 250 μlethanol for the precipitation of the single-stranded phage DNA. Afterincubation for 10 min at room temperature, the solution was centrifugedfor 10 min at 15,000 rpm. The supernatant was discarded and the pelletwashed in 70% ethanol and dried briefly under vacuum. The pellet wassuspended in 10 μl distilled water containing the sequencing primer forautomated sequencing of the peptide insert (BigDye Terminator bytesequencing with standard M13-40 primer on an Applied Biosystem 877/377).The remaining single phage solution was used for ELISA experiments.

2) Results

After three rounds of biopanning, phages presenting the followingsequences were isolated (Table I, II, III and IV).

TABLE I Linear peptides sequences isolated from 17 different phageseluted with in 1 mM colominic acid SEQ ID NO: sequence 5 SWLQMPWALVRT 5SWLQMPWALVRT 4 TRLAPLVFPLDY 6 EIHLRMIKQITI 7 WHLEYMWRWPRL 5 SWLQMPWALVRT5 SWLQMPWALVRT 8 LIEQRLPKHILT 9 YETSSSRLLAYA 5 SWLQMPWALVRT 10TLASQLSNTSAY 5 SWLQMPWALVRT 11 SDQGVNGSWSNP 4 TRLAPLVFPLDY 5TRLAPLVFPLDY 5 SWLQMPWALVRT 4 TRLAPLVFPLDY

TABLE II Linear peptides sequences isolated from 20 different phageseluted with 0.2 M glycine. SEQ ID NO: Sequence 1 DSPLVPFIDFHP 2LWQPPLLIPGIDF 2 LWQPPLLIPGIDF 12 WHNWNLWAPASPT 3 QIEPWFTPEDFP 1DSPLVPFIDFHP 3 QIEPWFTPEDFP 13 WHWQWTPWSIQP 2 LWQPPLIPGIDF 5SWLQMPWALVRT 2 LWQPPLIPGIDF 1 DSPLVPFIDFHP 15 SHLDLSTGHRTS 1DSPLVPFIDFHP 1 DSPLVPFIDFHP 5 SWLQMPWALVRT 1 DSPLVPFIDFHP 14IKSPLTWLVPPD 1 DSPLVPFIDFHP 1 DSPLVPFIDFHP

5 linear peptides have a high occurrence in the phages isolated afterthree rounds of biopanning (Table III).

TABLE III Alignment of the 5 more frequent linear sequences SEQ Fre- IDquence NO: Sequence 9/40 5 S W L Q M P W A L V R T 8/40 1 D S P L V P FI D F H P 4/40 4 T R L A P L V F P L D Y 4/40 2 L W Q P P L I I G I D F2/40 3 Q I E P W F T P E D F P

An alignment at GeneStream Align Home page to sequence SEQ ID NO: 1,showed that the sequence similarity varies from 42.9% for SEQ ID NO: 2,30.8% for SEQ ID NO: 3, 28.6% for SEQ ID NO: 4, to 8.3% for SEQ ID NO:5.

34 phage clones displaying cyclic peptides bound the antibody in a dosedependent manner after three cycles of biopanning, and they did not bindto an irrelevant antiboty of the same isotype. DNA from 16 of theseclones showing the highest value in the ELISA test were prepared andsequenced (Table IV). 3 clones exhibited the same sequence (SEQ ID NO:17) and the dimeric motif WP was found in 5 clones.

TABLE IV Cyclic peptides sequences isolated from phages SEQ ID NO:Peptide n° Sequence SEQ ID NO: 16 2 CYPLNPEVYHCG SEQ ID NO: 17 3CWPLSHSVIVCG SEQ ID NO: 17 5 CWPLSHSVIVCG SEQ ID NO: 18 6 (p65)CSSVTAWTTGCG SEQ ID NO: 19 8 CYMASGVFLCG SEQ ID NO: 17 9 CWPLSHSVIVCGSEQ ID NO: 20 10 CWPLGPSTYICG SEQ ID NO: 21 11 (p66) CSLIASMETGCG SEQ IDNO: 22 CSKIASMETGCG SEQ ID NO: 16 12 CYPLNPEVYHCG SEQ ID NO: 23 13CYIGDPPFNPCG SEQ ID NO: 24 14 CWPLGDSTVICG SEQ ID NO: 25 15 CPLRLAFTFGCGSEQ ID NO: 26 16 CTRMSHGYWICG

EXAMPLE 2 Analysis of Peptides Specificity by Competitive Phage ELISA

1) Materials and Methods

1.1) Materials

-   -   anti-PSA monoclonal antibody 735 or 30H12    -   MaxiSorp™ plates (NUNC) for antibody coating: ELISA plates    -   microtiter plates (NUNC) for phage dilution: dilution plates    -   M13 bacteriophages presenting peptides of Tables III and IV from        example 1    -   HRP conjugated anti-M13 antibody (PHARMACIA 27-9411-01)    -   ABTS [2,2′Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),        SIGMA]    -   Colominic acid (SIGMA)    -   Dextran (SIGMA)

1.2) Buffers

-   -   PBS pH 7.4    -   Blocking solution: 0.5% BSA in PBS    -   TBS    -   TBST: TBS containing 0.05% Tween 20    -   Horse Radish Peroxydase-conjugated anti-M13 antibody solution:        1/5000 in TBST    -   Horse Radish Peroxydase (HRP) substrate solution: 22 mg ABTS in        100 μl 50 mM sodium citrate pH 4.0. Prior enzymatic reaction,        add 36 ml 30% H₂O₂ to 21 ml ABTS solution.    -   competitor solution: 1 mM colominic acid in TBST    -   control solution: 1 mM dextran in TBST

1.3) Method

MaxiSorp™ plate wells were coated with 100 μl mAb solution (25 μg/ml)for 2 h at room temperature. The control wells were coated only with theblocking solution. In parallel, the phage dilution plates were blockedwith 200 μl blocking solution for 2 h. The antibody coated wells and thecontrol wells of the ELISA plates were blocked with 200 μl blockingsolution for 1 h. In parallel the phage dilution plates were washed 6times with TBST and 120 μl TBST was added to the wells. An appropriatevolume of a phage solution was added to the first well and the volumewas adjusted to 140 μl with TBST. The phage solution in well one wasdiluted in the ratio 1/7 by taking 20 μl out of the first well andtransferring into the second, also to achieving the total volume of 140μl. This was repeated for the remaining wells. The phage dilutions forthe control wells were done the in same way. The blocked ELISA plate waswashed 6 times with TBST and the phage dilutions or the competitorsolution were added. After incubation for 1 h, the plates were washed 10times with TBST. After incubation for 1 h, the wells were washed 10times with TBST. 100 μl of the HRP-conjugated M13 antibody solution wasadded to the wells. After incubation for 1 h, the wells were washed 10times with TBST and 100 μl of the HRP substrate solution (with H₂O₂) wasadded to the wells. The plates were read at 405 nm using a microplatereader.

2) Results

The binding specificity of peptides SEQ ID NO: 1 to 5 from example I wastested in a competitive ELISA using colominic acid as competitor. Theresults presented in Tables V to DC are expressed as percentage ofbinding, by comparison with phage presenting SEQ ID NO: 5 at aconcentration of 710 ng/well (100%)

TABLEAU V Binding of phage presenting SEQ ID NO: 5 to mAb 735 Phageconcentration (ng/well) 710 109 18 3 1 0 0 phage 100 ± 3 102 ± 0 98 ± 073 ± 4 18 ± 2 4 ± 3 2 ± 1 phage + 106 ± 0 103 ± 5 90 ± 0 36 ± 0  5 ± 0 1± 0 1 ± 0 colominic acid phage + 102 ± 2 103 ± 1 97 ± 1 62 ± 1 14 ± 0 5± 0 1 ± 0 dextran phage +  5 ± 1  3 ± 1  1 ± 0  1 ± 0  0 ± 0 0 ± 0 0 ± 0BSA

TABLEAU VI Binding of phage presenting SEQ ID NO: 1 to mAb 735 Phageconcentration (ng/well) 1003 154 26 4 1 0 0 phage + mAb735 108 ± 2 111 ±7 111 ± 3 112 ± 12 86 ± 15 28 ± 2  7 ± 0 phage + colominic 108 ± 0  109± 12  90 ± 3 42 ± 3 12 ± 1  2 ± 1 0 ± 0 acid + mAb735 phage + dextran +109 ± 6 110 ± 6 112 ± 5 107 ± 1  77 ± 4  24 ± 4  8 ± 0 mAb735 phage +BSA  10 ± 1  4 ± 1  3 ± 0  1 ± 1 0 ± 0 0 ± 0 0 ± 0

TABLEAU VII Binding of phage presenting SEQ ID NO: 4 to mAb 735 Phageconcentration (ng/well) 870 134 22 4 1 0 0 phage + mAb735 107 ± 7   112± 10 109 ± 1 108 ± 2 67 ± 2  17 ± 0  4 ± 0 phage + colominic 96 ± 7 106± 3  89 ± 1  29 ± 1 5 ± 0 1 ± 0 0 ± 0 acid + mAb735 phage + dextran + 96 ± 12 108 ± 7 113 ± 0 105 ± 0 66 ± 3  16 ± 1  5 ± 0 mAb735 phage +BSA 10 ± 1  4 ± 1  1 ± 1  0 ± 0 0 ± 0 0 ± 0 0 ± 0

TABLEAU VIII Binding of phage presenting SEQ ID NO: 2 to mAb 735 Phageconcentration (ng/well) 796 122 20 3 1 0 0 phage + mAb735 108 ± 5 109 ±3  112 ± 10 89 ± 11 27 ± 4 4 ± 2 2 ± 1 phage + colominic 109 ± 6  114 ±12 100 ± 6 48 ± 6  10 ± 1 1 ± 0 1 ± 0 acid + mAb735 phage + dextran +109 ± 8 111 ± 6 110 ± 5 99 ± 1  27 ± 1 5 ± 1 1 ± 0 mAb735 phage + BSA 12 ± 1  9 ± 1  4 ± 3 2 ± 1  1 ± 0 0 ± 0 0 ± 0

TABLEAU IX Binding of phage presenting SEQ ID NO: 3 to mAb 735 Phageconcentration (ng/well) 859 132 22 4 1 0 0 phage + mAb735 111 ± 8  109 ±16 114 ± 8 99 ± 10 50 ± 1  13 ± 1  6 ± 0 phage + colominic 104 ± 2 107 ±2  85 ± 7 27 ± 3  4 ± 1 1 ± 1 0 ± 0 acid + mAb735 phage + dextran + 109± 2 108 ± 0 108 ± 3 107 ± 8  57 ± 3  13 ± 1  6 ± 0 mAb735 phage + BSA 10 ± 0  5 ± 1  2 ± 1 1 ± 0 0 ± 0 0 ± 0 0 ± 0

The phage presenting sequence SEQ ID NO: 1 (DSPLVPFIDFHP) showed thebest binding to mAb 735 in comparison with the other phages. Thisbinding was competed with colominic acid, whereas dextran showed nocompetition effects. The phage presenting sequence SEQ ID NO: 4 showedsimilar values. Because of the low occurrence of sequence SEQ ID NO: 4it was decided to synthesize sequence SEQ ID NO: 1 (peptide p21) and arandomized variant of sequence SEQ ID NO: 1 (peptide p22: PDHIFVFSPDLP,SEQ ID NO: 28) as control.

The phages presenting cyclic peptides corresponding to sequenceCSSVTAWTTGCG (SEQ ID NO: 18) and CSKIASMETGCG (SEQ ID NO: 22)respectively, in which the two cysteine residues are linked via adisulfide-bridge, showed the best binding to mAb 30H12 in comparisonwith the other phages. Thus, it was decided to synthesize thecorresponding cyclic peptides (p65 and p66).

EXAMPLE 3 Analysis of p21, p65 and p66 Specificity by CompetitivePeptide ELISA, ELISA and Competitive Binding To Psa-Ncam ExpressingCells

1) Preparation of Biotinylated Peptide-BSA Conjugates

1.1) Materials

-   -   m-maleimidobenzoyl-N-hydroxysuccimide ester (MBS, SIGMA M2786,        PIERCE 1) 20311)    -   biotinamidocaproate-N-hydroxysuccimide ester (NHS-Biotin, SIGMA        02643, PIERCE 20217)    -   dimethylformamide (DMF, SIGMA)    -   BSA (CALBIOCHEM 122605)    -   cysteine-containing synthetic peptides deriving from p21        (DSPLVPFIDFHPC, SEQ ID NO: 29), p65 and p66 and corresponding        cysteine-containing control peptides deriving from p22        (PDHIFVFSPDLPC, SEQ ID NO: 30), p65 and p66 reverse peptides,        respectively.    -   PD-10 size exclusion column (AMERSHAM-PHARMACIA 17-0851-01)    -   Ultrafree-4-centrifuge filters & tub Biomax 50K NMWL membrane, 4        ml volume (MILLIPORE UFV4BQK25)    -   L-cysteine (SIGMA)

1.2) Buffers

-   -   MBS stock solution: 13 mg/ml in DMF    -   NHS-Biotin stock solution: 2.5 mg/ml in DMF (These        concentrations lead to highly activated BSA molecules with        approximately 5 molecules biotin/BSA molecule)    -   conjugation buffer: 0.083 M NaH₂PO₄, 0.9 M NaCl , pH 7.2.    -   PBS pH 7.4,    -   cysteine solution: L-cysteine 100 mg/ml in conjugation buffer

1.3) Method

10 mg BSA were dissolved in 2 ml conjugation buffer and 140 μl of theMBS/NHS-Biotin stock solution were added. The solution was incubated for1 h at room temperature, with gentle agitation. The PD-10 column wasequilibrated with 50 ml conjugation buffer. After adding 2.14 ml of thesolution onto the column, the activated BSA was eluted with 0.5 mlaliquots of conjugation buffer. The protein elution was monitored at 280nm. An appropriate amount of the cysteine containing peptide wasdissolved in 1 ml conjugation buffer. For a 5 mer (MW 1500, 1.14 mgpeptide was added to 10 mg BSA). This peptide solution was added to thepooled fractions containing maleimide-activated/biotinylated BSA. After2 h incubation at room temperature, 100 ml cysteine solution was addedto block the non-reacted maleimide groups. After 1 h, the reactionsolution was dialysed 5 times with 1 ml PBS, in an ultrafiltration unit.The biotinylated conjugate was dissolved in PBS and aliquots stored at−20° C.

2) Competitive ELISA of Biotinylated Peptide-BSA Conjugates on Anti-PSAmAb

2.1) Materials, Buffers and Method

2.1.1) Materials

-   -   mAb 735 and 30H12    -   MaxiSorp™ plates (NUNC) for coating the antibody: ELISA plates    -   microtiter plates (NUNC) for peptide dilution: dilution plates    -   Biotinylated peptide-BSA conjugates prepared as above described    -   Extravidin alkaline phosphatase-avidin conjugate (SIGMA E2636)    -   p-nitrophenyl-phosphatase alkaline substrate (SIGMA 104-105)        2.1.2) Buffers    -   PBS pH 7.4    -   Blocking solution: 0.5% BSA in PBS    -   TBS    -   TBST: TBS containing 0.05% Tween 20    -   Extravidin alkaline phosphatase-avidin conjugate solution:        1/5000 in TBST    -   Alkaline phosphatase substrate solution; 1 tablet in 5 ml 50 mM        NaHCO₃, 1 mM MgCl₂ solution, pH 9.6.    -   mixed competitor solution: 1 mM colominic acid with a        peptide-BSA conjugate gradient in TBST        2.1.3) Method

Maxisorp™ plate wells were coated with 100 μl mAb solution (25 μg/ml)for 2 h at room temperature. The control wells of the ELISA plates wereonly coated with the blocking solution. In parallel, the peptide-BSAconjugate dilution plates were blocked with 200 μl blocking solution for2 h. The antibody coated wells and the control wells were blocked with200 μl blocking solution for 1 h. In parallel the peptide-BSA conjugatedilution plates were washed 6 times with TBST and 120 μl TBST was addedto the wells. An appropriate volume of peptide-BSA conjugate solutionwas added to the first well and the volume adjusted to 140 μl with TBST.The peptide-BSA conjugate in well one was diluted in the ratio 1/7 bytaking 20 μl out of the first well and transferring into the second alsoto achieve the total volume of 140 μl. This was repeated for theremaining wells. The peptide-BSA conjugate dilutions for the controlwells were done the same way. The blocked ELISA plates were washed 6times with TBST and the peptide-BSA conjugate dilutions or 100 μl of themixed competitor solution was added to the wells. After incubation for 1h, the wells were washed 10 times with TBST and 100 μl of the alkalinephosphatase substrate solution was added to the wells. The plates wereread out after 10 to 60 min using a microplate reader at 405 nm.

2.2) Results

The specificity of peptide p21 (SEQ ID NO: 1) was investigated in acompetition ELISA using biotinylated p21-BSA conjugate and a randomizedvariant of p21 (p22) conjugate as a control. The results presented inTables X and XI are expressed as percentage of binding by comparisonwith peptide p21 at the highest concentration (9.45 10-5 M) whichcorresponds to 100% binding.

TABLE X Binding of p21 to mAb 735 in competitive ELISA Peptideconcentration (M) 9.45 1.67 2.94 5.20 9.27 1.62 2.86 5.04 8.89 10⁻⁵ 10⁻⁵10⁻⁶ 10⁻⁷ 10⁻⁸ 10⁻⁹ 10⁻⁹ 10⁻¹⁰ 10⁻¹¹ Peptide + mAb 735 100 ± 11 93 ± 1095 ± 1 91 ± 2 93 ± 9  56 ± 4  13 ± 1  2 ± 0 0 ± 0 Peptide + colominic 43± 3 34 ± 3  23 ± 1 15 ± 3 5 ± 2 0 ± 1 0 ± 0 0 ± 0 0 ± 0 acid + mAb 735Peptide + chondroitin 96 ± 8 99 ± 4  99 ± 8 94 ± 7 92 ± 2  50 ± 1  11 ±2  3 ± 0 2 ± 0 sulfate C + mAb 735 Peptide + BSA  3 ± 1 1 ± 0  0 ± 0  0± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0

TABLE XI Binding of p22 to mAb 735 in competitive ELISA Peptideconcentration (M) 9.45 1.67 2.94 5.20 9.27 1.62 2.86 5.04 8.89 10⁻⁵ 10⁻⁵10⁻⁶ 10⁻⁷ 10⁻⁸ 10⁻⁹ 10⁻⁹ 10⁻¹⁰ 10⁻¹¹ Peptide + mAb 735 32 ± 1 9 ± 0 1 ±0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Peptide + colominic 27 ± 0 5 ± 0 0± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0 ± 0 acid + mAb 735 Peptide +chondroitin 29 ± 0 8 ± 1 3 ± 1 2 ± 0 2 ± 1 2 ± 0 2 ± 0 2 ± 0 2 ± 0sulfate C + mAb 735 Peptide + BSA  3 ± 1 1 ± 1 0 ± 0 0 ± 0 0 ± 0 0 ± 0 0± 0 0 ± 0 0 ± 0

Table X shows a clear inhibition of p21 binding in the presence ofcolominic acid. By comparison, chondroitin sulfate C had no influence onthe binding of peptide B. Table XI shows no binding to mAb 735 for therandomized variant of p21 conjugate (p22); no differences were observedwhen colominic acid or chondroitic sulfate C were present. These resultslead to the conclusion that sequence SEQ ID NO: 1 (peptide p21) bindsspecifically to mAb 735 in a concentration-dependent manner.

3) ELISA with Peptide-BSA Conjugate

The specificity of the cyclic peptides was investigated in an ELISAassay using plates coated with 30H12 antibody, following the protocol asdescribed above for the competitive ELISA, with the exception that thecompetitor was omitted. The results presented in FIG. 12A demonstratethat the cyclic peptides bind to the antibody in an antigen-specificmanner. Peptides p65 and p66 exhibiting the highest binding were chosenfor further studies.

4) Competitive Binding to PSA-NCAM Expressing Cells

The specificity of p65 and p66 peptides was tested in a competitionassay using anti-PSA antibodies and PSA-NCAM expressing cells.Pre-incubation of the 30H12 monoclonal antibody with 0.1 mM of eitherp65 or p66 peptide prevented its binding to PSA-NCAM expressing cells(FIG. 12D versus 12B). The binding specificity was examined in greaterdetail by testing peptide recognition by another anti-PSA mononclonalantibody (MenB). The results presented in FIG. 12C demonstrate that p65and p66 peptides bind only to 30H12; pre-incubation of the peptides withMenB did not prevent recognition of PSA-NCAM (FIG. 12 C). Thus, p65 andp66 mimotopes appear to be specific for a unique (idiotypic)determinant.

EXAMPLE 8 Analysis of PSA Mimetic Peptides Bioactivity

1) Materials and Methods

1.1) Animals

GFP transgenic mice have been previously described in Hadjantonakis etal. (Biotechnol., 2002, 2, 11-), and all analysis was performed on Swissbackground. NCAM knock out mice (NCAM −/−) have been previouslydescribed in Cremer et al., precited.

1.2) Dorsal Root Ganglions (DRG) Explant Culture

E13.5 DRGs were dissected out from mice embryos in HBSS medium andseeded on glass coverslips coated with polylysine or with peptideslinked to BSA. Explants were cultured in the presence or the absence ofthe peptides under soluble form (40 μM) in two ml of neurobasal medium(DMEM/Ham's F12, 3:1 (V/V), GIBCO, buffered with 20 mM Hepes),supplemented as described in Faivre-Sarrailh et al., J. Cell. Sci.,1999, 18, 3015-3027 and Chazal et al., J.Neurosci., 2000, 20, 1446-1457.

1.3) Subventricular Zone (SVZ) Explant Culture.

Cultures of SVZ explants were performed as described in Chazal et al.,precited. Briefly, 1-day-old mice were killed by rapid decapitation.Brains were dissected out and sectionned by Vibratome (Leica). The SVZfrom the lateral wall of the anterior lateral ventricle horn wasdissected out in HBSS medium (LIFE TECHNOLOGIES) and cut into 200-300 μmdiameter explants. The explants were mixed with Matrigel (BECKTONDICKINSON) and cultured in four-well dishes. After polymerisation, thegel was overlaid with 400 μl of serum-free medium containing B-27supplement (LIFE TECHNOLOGIES), in the presence or the absence of 40 μMof peptides (p65, p66, reverse p65, p21 or p22) and 70 U of Endo N permilliliter.

1.4) Immunohistochemistry

Fixed DRGs (Dorsal Root ganglia) explants and sections were incubated at4° C. respectively 2 hr with an anti-neurofilament (SMI-31, dilution1:800, STERNBERGER MONOCLONALS) and overnight with an anti-PSA antibody(dilution 1:200, Rougon et al, J. Cell. Biol., 1986, 103, 2429-2437).Revelation was performed by 1 hr incubation with the correspondingfluorescent-labeled secondary antibody (Goat anti-mouse IgM or IgGconjugated with texas red, IMMUNOTECH)

1.5) Cell Migration Distance (SVZ Explants) and Neuronal Outgrowth (DRGExplants).

After 48 h in culture, explants were examining directly (SVZ) or afterovernight fixation with a 4% paraformaldehyde solution in PBS andimmunostaining (DRGs). Observation was done using 2, 5×, 5×, 10× and 32×objectives (Axiovert 35M, ZEISS). Images were collected with a videocamera (Cool View, PHOTONIC SCIENCE) and analysed using image-processingsoftware (Visiolab1000, BIOCOM). Mean migration distance (calculated onfive different experiments, including at least five explants percondition) or mean length of the longest neurite (calculated on twodifferent experiments, including at least eight explants per condition),was the distance in micrometers between the explant edge and the borderof the cell migration front. Four measurements were performed for eachexplant. The significance of the differences between the control and thedifferent experimental conditions was calculated by Student's t test.

1.6) Transplantation

100 μm diameter explants of 1-day-old GFP mice SVZ were incubatedfifteen minutes in DMEM supplemented with 10% fetal bovine serum inpresence of 0.01 M of p65 or reverse peptide and stereotaxically grafted(0.5 μl) into six weeks old mice SVZ as described in Lois andAlvarez-Buylla (Science, 1994, 264, 1145-1 148). Three or four daysafter the graft, animals were perfused intracardiacally with a 4%paraformaldehyde solution in PBS. Brains were dissected out, postfixed,cryoprotected, and freezed in isopentane. Sagittal serial sections (12μm) were cut with a Leica microtome and immunostained as describedabove. GFP cells arriving in the olfactory bulb after three days wereobserved using UV fluorescence with a 40× objective (Axioscope, ZEISS)and counted on two different experiments (four animals per condition).The significance of the difference between the two conditions wascalvalated by Student's t test.

1.7) Intravitreal Injections and Retinal Whole Mount.

Injections were performed as described in Monnier et al. (DevelopmentalBiology, 2001, 229, 1-14). Briefly, a 2×2 cm window was cut into theshell over E3 chick embryos. One μl of Fast green with 10 mM of p65 orreverse peptide was injected into the right eye vitreous body using acapillary. After a five-days incubation at 37° C., (E8) retinae weredissected, spread onto nitrocellulose filters (MILIPORE), and fixed with4% paraformaldehyde solution in PBS. Two small DiI(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)crystals were applied dorsally to the optic fissure. Retinae were storedin the dark at 37° C. for 10 days until the dye reached the axonalgrowth cones in the fissure, mounted in glycerol:PBS (9:1, v:v) andanalysed using confocal microscopy.

2) Results

2.1) Effect of Psa Mimetic Peptides on Axonal Growth and FasciculationIn Vitro (FIG. 2)

Mouse dorsal root ganglion explants (E13.5) were cultured in thepresence of p65 and p66 cyclic mimetic peptides, either in soluble formor coated on the microplates as BSA conjugates. Cells cultured in theabsence of peptides or in the presence of reverse peptides (coated or insoluble form) were used as controls.

The effect of the peptides on neurite outgrowth and fasciculation wasanalysed qualitatively and quantitatively (FIG. 2 A to H).

p65 and p66, in a soluble form, induce a clear defasciculation of axonbundles (C versus A and F versus D) and a significant increase in axonalgrowth (C versus A) by comparison with the controls.

Interestingly, p65 and p66, in a coated form, induced an opposite effecton fasciculation (B versus A and E versus D) and no effect on neuriteoutgrowth.

These results were confirmed by quantitative analysis showing that p65and p66, in soluble form, increased neurite outgrowth by 34% and 21%respectively, compared to the controls, whereas the same peptides in thecoated form induce no significant increase on neurite outgrowth (FIGS.2G and 2H).

These results demonstrate that PSA mimetic peptides are able to modulateaxonal growth and fasciculation in vitro.

2.2) Effect of PSA Mimetic Peptides on Axonal Fasciculation and GuidanceIn Vivo (FIG. 3)

p65 and p66 were injected into chicken embryo eyes (E3) and the retinawere observed at E9. The results presented in FIG. 3 show that thepresence of PSA mimetic peptides during retina development induce axonsguidance and fasciculation defects; axons leave their fascicle and runperpendicularly to it (arrowheads in D, G, E and H). By comparison, nodefect in axon guidance and fasciculation is observed after injection ofthe control peptides.

These results demonstrate that PSA mimetic peptides are able to modulateaxonal growth and guidance in vivo.

2.3) Effect of PSA Mimetic Peptides on Cell Migration In Vitro (FIGS. 4to 7)

The effect of PSA mimetic peptides on cell migration in vitro wasanalysed on subventricular explants from 1-d-old normal mice (NCAM +/+),heterozygous (NCAM +/−) or knock out mice (NCAM −/−) cultured inMatrigel, in presence of the mimetic peptides (p65, p21, p66), differentform of p65 peptide (cyclic, linear, linear and acetylated), the controlpeptides (reverse p65 and reverse p66 and p22) or endosialidase N. Theresults are illustrated in FIGS. 4 to 7.

-   -   FIG. 4 (A to I) show that the addition of p65 and p66 peptides        to the culture increases the rate of migration of the neuronal        precursors and a modification of their chain-like arrangement (C        and F). These effects were not found with the reverse peptides        (A and D) and were PSA-dependent since they were abolished by        endo N treatment (B and E).

These results were confirmed by the quantitative analysis (G and H)demonstrating that p65 and p66 induce a significant increase in the rateof migration of the neuronal precursors (+40% and +26% respectively at0.4 μM, compared with the control without peptide), whereas endo Ndecreases it (−21%, compared with the control without peptide).

Dose-response curve of p65 (1) shows that the optimal effect on cellmigration is observed starting from and above 0.4 μM peptide.

-   -   FIG. 5 show that cyclisation of p65 is a prerequisite to p65        promoting effect since the corresponding amino acid sequences in        a linear form, either N-acetylated or not, are unable to        stimulate cell migration.    -   FIG. 6 show that p65 effect is dependant upon PSA expression        since a significant reduction of the precursor migration was        observed in the NCAM −/− mice by comparison to the NCAM +/−mice,        in the presence of reverse peptide or p65 peptide; the effect        were comparable in endo N treated NCAM +/−mice and NCAM +/− mice        and the p65 did not reverse the impaired migration in the NCAM        −/− mice.    -   FIG. 7 show that p65 induce a significant increase in the rate        of migration of the neuronal precursors, compared to the        corresponding control peptide transverse p65).

By contrast, p21 induces a significant decrease in the rate of migrationof the neuronal precursors, compared to the corresponding controlpeptide (p22 peptide); the decrease is comparable to that observed inthe endo N treated cells.

These results demonstrate that the mimetic peptides are able tostimulate (p65) or inhibit (p21) cell migration in a PSA-dependantmanner.

2.4) Effect of PSA Mimetic Peptides on Cell Migration in vivo (FIG. 8)

The effect of PSA mimetic peptides on cell migration in vivo wasanalysed by grafting tissue and evaluating migration of SVZ cells. Smallpieces of SVZ tissue (100 μm diameter) from 1-day-old GFP mice weregrafted in the SVZ area of adult mice, in the presence of p65, p65reverse peptide or in the absence of peptide. The results areillustrated in FIG. 8.

FIG. 8 show that the presence of p65 peptide increases significantly thenumber of GFP positive cells migrating to the olfactory bulb (via theRostral Migration Stream or RMS), by comparison with the control. Thiseffect was observed as early as 3 days post-engraftment (FIG. 8E). Theseresults were confirmed by the quantitative analysis showing that thenumber of GFP positive cells present in the olfactory bulb 3 days afterthe graft is increased 17 times in the presence of p65, by comparisonwith the control (FIG. 8G).

These effects were PSA-dependent since they were abolished in NCAMknock-out mice.

These results demonstrate that the mimetic peptides are able to increasethe migration of PSA positive cells.

EXAMPLE 9 Analysis of Functional Recovery from Spinal Cord Injury afterInjection of PSA Mimetic Peptides

1) Material and Methods

1.1) Spinal Cord Surgery and Peptide Delivery

Male Swiss-CD1 mice (8-10 week-old) were anesthetized with a mixture ofketamine and xylazine. The spinal cord had been exposed by making amidline incision of the skin, and by retracting the paravertebralmuscles. A laminectomy was performed at the T7-T8 level and the spinalcord exposed. Using iridectomy scissors, a bilateral dorsal hemisection,transecting left and right dorsal finiculus, the dorsal horns, butsparing most parts of the ventral fimiculus, was performed, resulting ina complete transection of the dorsomedial main Cortico Spinal Tract(CST). For the series of mice receiving the peptides a Surgicoll pledgetsaturated with 10 μl of either p65 or reverse p65 (10 μM) peptides wasapplied over the transection site and covered with petroleum jelly toprevent diffusion. All inside muscle layer were sutured using finethread. Skin was sutured using surgical staples. Following surgery, 1 mlof saline was administrated subcutaneously to prevent dehydratation, andthe mice were placed under a heating lamp until they had recovered fullyfrom the narcosis. The mice were then returned to their home cage andreceived a daily subcutaneous injection of antibiotic Baitryl™ toprevent infection. Manual bladder evacuation was made until recovery ofcomplete autonomic bladder function.

1.2) Functional Testing (FIG. 9)

Functional evaluation of the animals was performed at day (D) D1, D4,D7, D14, D21, D28, D35, during the first week following the spinal cordlesion and then on a weekly basis until D35, by two different observersblinded to group identity. Locomotor recovery was evaluated using theBBB test (Basso, Beattie and Bresnahan test; Basso et al., Restor.Neurol. Neurosci, 2002, 5, 189-218). The scale ranges from 0 (noobservable hindlimb movement) to 21 (normal gait) and can be subdividedinto three ranges. Scores from 0 to 7 correspond to a low recovery(movements of joints, no weight-support, no paw placement). Scores from8 to 13 can be related to an intermediate recovery (paw placement,forelimb-hindlimb coordination). Scores from 14 to 21 can be related toa very good recovery. Finally on the last day of test (D35), mice weresubjected to the rotarod test to assess fine motor coordination.

1.3) Histology (FIG. 9)

At D5, lesioned animals which received either p65 (n=3) or reversed p65(n=3) were transcardiacally perfused. Spinal cords were sectioned in thesagittal plane at 20 μm intervals in blocks of 10 mm length at thelesion site. To examine lesion extent, Nissl staining was performed inseries of 1-in-6 sections in all animals. Series of 1-in-3 sections werestained with the MenB anti-PSA (mouse IgM) and/or anti-GFAP (mouse IgG)antibodies. Bound antibodies were revealed by the appropriatefluorescently labelled secondary antibodies.

2) Results

After severed lesions, brain and spinal axons do not advance through theadult CNS. Instead, these fibres are trapped at the site of damage andremain disconnected from synaptic targets, leading to profound andpersistent deficits in many clinical cases. Spinal cord injury (SCI) isthe clearest example of a condition in which axonal disconnection leadsto significant disability despite minimal neuronal death.

Thus, the effect of the p65 PSA mimetic peptides (and its reversecounterpart, taken as control) on functional recovery from spinal cordinjury was analyzed in mice.

2.1) Improved Locomotor Recovery after Spinal Cord Injury in Mice whichReceived p65 Peptide at the Site of Lesion

The results presented in FIG. 10 (A and B) demonstrate that PSA mimeticpeptide treatment is correlated with functional recovery aftermidthoracic dorsal hemisection injury. More precisely, recovery wasassessed using a standardized open-field measure of locomotor functionafter spinal cord injury, the BBB score. In this scale, 21 is normalfunction and 0 is bilateral total paralysis of the hind limbs. All micehad scores of 0 at D1 post injury. The p65-treated mice graduallyrecovered partial function over a 45 day observation period (FIG. 10A).The scores of p65-treated mice were significantly higher thanp65-reverse controls starting from D14 post-injury and throughout thefollowing time-points. Considering the time-period (14 days) when thisimprovement is observed it is compatible with some long-distance growthof CST fibers extending from the lesion site to the lumbar motor pool.Local sprouting in the lumbar cord as well as rearrangements of otherdescending tracts, such as the rubrospinal system, or of distalintrinsic spinal cord circuitry might also contribute. Regardless ofmechanism, the locomotor recovery in the p65-treated mice wassignificantly greater than in control animals. The p65 beneficial effecton recovery was further assessed by a rotarod test performed at D35(FIG. 10B).

2.2) Decrease of Reactive Gliosis at D5

To assess the effect of the peptide, quantification of the GlialFibrillary acidic Protein (GFAP) expression as an index of reactivegliosis, was undertaken in a subset of animals taken D5 followingsurgery. These animals were selected in a blinded manner forquantification. 3 p65 (10 slices per animal) and 3 p65-reverse (10slices per animal) animals were analyzed. Double-labelling was performedwith MenB anti-PSA antibody. Quantification was also performed in ablinded manner.

A significant difference between p65 treated and p65-reverse animals wasobserved (FIGS. 11A and B), demonstrating that p65-treatment reducedreactive gliosis by 40% compared with the reverse p65-treatment,possibly by preventing migration inside the scar or by inhibiting otherprocesses involved such as action of inflammatory cytokines. In any casethese results supported the fact that functional recovery was better inp65-treated mice.

The invention claimed is:
 1. A cyclic peptide comprising SEQ ID NO:18,wherein the side chain of the cysteine residue at position 1 of SEQ IDNO:18 is covalently attached to the side chain residue of the cysteineat position 11 via a disulfide bond.
 2. The cyclic peptide of claim 1,which consists of SEQ ID NO:18.
 3. A composition comprising the cyclicpeptide of claim 1 and a pharmaceutically acceptable carrier.
 4. Amethod of preparing a pharmaceutical composition, comprising combiningthe cyclic peptide of claim 1 and a pharmaceutically acceptable carrierto form the pharmaceutical composition.